Pasteurella multocida is a gram-negative, non-motile, rod shaped, facultative anaerobe which is isolated from a wide range of animals and birds from all over the world.
The P. multocida isolates are classified into five serogroups (A, B, D, E and F) based on capsular antigens and 16 serotypes by somatic antigens (Rimler and Rhoades, 1989). Serogroup A is most commonly associated with fowl cholera in birds followed by serogroup D (Rhoades and Rimler, 1989). Among the isolates, serogroup F strains are predominantly isolated from poultry and turkeys, but rarely from calves (Shewen and Conlon, 1993; Catry et al., 2005). In pigs, atrophic rhinitis and pneumonia are primarily associated with serogroups D and A which express dermonecrotizing toxin (Dungworth, 1985). On the other hand P. multocida serogroups B and E are usually associated with hemorrhagic septicemia in cattle and water buffaloes in tropical and sub-tropical regions of Africa and Asia (Carter and de Alwis, 1989; Rimler and Rhoades, 1989; Shewen and Conlon, 1993). In contrast P. multocida serogroups B and E are rarely isolated North America cattle population (Confer, 1993). More than 92% of P. multocida isolated from the US cattle which cause severe suppurative bronchopneumonia belong to serotype A:3 (Ewers et al., 2006; Confer et al., 1996; Weekley et al., 1998). P. multocida infection in calves results in significant production yield losses and mortality (Ewers et al., 2006; Confer et al., 1996; Dalgleish, 1989; Weekley et al., 1998). Furthermore, P. multocida is often associated with bovine respiratory disease complex (BRDC) along with Mannheima haemolytica and Histophilus somni. From 2001, bovine pneumonic pasteurellosis due to P. multocida infection has increased in the UK cattle population. In many UK cases, P. multocida infections exceeded the number of outbreaks caused by M. haemolytica induced bovine bacterial pneumonia (Veterinary Laboratories Agency, 2007). Worldwide, P. multocida serogroup A isolates are one of the major pathogens associated with BRDC (Frank, 1989; Rimler and Rhoades, 1989).
P. multocida isolates associated with BRDC have numerous virulence or potential virulence and virulence-associated factors like adhesins and filamentous hemagglutinin which aid in adherence and colonization, iron acquisition proteins and transport systems, extracellular enzymes such as neuraminidase, endotoxin (lipopolysaccharide, LPS), polysaccharide capsule and a variety of outer membrane proteins (OMPs). Immunity of cattle against respiratory pasteurellosis is poorly understood; however some reports indicate that high serum antibodies against P. multocida OMPs are important for enhancing resistance against this bacterium.
There are a few commercial vaccines currently available against P. multocida for use in cattle. These vaccines are predominately traditional bacterins and a live streptomycin-dependent mutant. However, the field efficacy of these vaccines is questionable and none of the vaccines afford reliable protection. Therefore, there remains a need for safe and effective vaccines to protect cattle against P. multocida infections.
State of the Art Review
Intervet (Merck Animal Health) makes a hyaE gene-deleted P. multocida vaccine. In contrast, the instantly disclosed P. multocida vaccine is hyaD gene deletion mutant. HyaD is a different gene in the same locus, and although both the gene deletions result in an acapsular phenotype, a skilled person could not have predicted ahead of this disclosure whether deleting the hyaD gene would result in a stable, viable acapsular phenotype. Moreover, according to U.S. Pat. No. 7,351,416 B2 (Examples 3 & 4), the ΔhyaE vaccine may be administered to steers (weighing about 500 pounds), or 2-3 month old calves (weighing over 150 pounds). In contrast, the target animal for the vaccines of the instant disclosure are calves as young as 4-6 weeks old, and weighing significantly less. Immune responses of very young animals are significantly different than older ones. Furthermore, vaccine safety is of paramount importance when used in young calves.
EP1831248B1 (to Intervet) describes a transposon generated mutant P. multocida, which is not directed or site specific. Bacteria harboring such transposon insertions are not likely to be approved by regulatory agencies for use in vaccines, and so disclosure of these types of mutations may fairly be viewed as preliminary work leading to targeted gene modification, including deletion. The mutant gene is reported as “ORF 15,” which is a membrane bound lysozyme inhibitor of c-type lysozyme. Finally, the vaccination challenge was done in poultry rather than calves.
WO2003086277A2 (to Merial) discloses attenuated P. multocida 1059. The gene deletions were initially produced using random signature tagged mutagenesis using transposon Tn5. Along with many specific and non-specific mutants, this library has mutants lacking PhyA, hyaC and hyaE genes which are involved with capsule biosynthesis. However, these mutants were generated by random mutagenesis and their genetic stability has not been tested over a long period. This is a critical property for a vaccine to be used as a modified live product under field conditions. Furthermore, P. multocida 1059 is an avian strain unsuitable for calf vaccination.